Plant virus-based vectors for expressing heterologous proteins in plants present promising biotechnological tools to supplement conventional breeding and transgenic technology. Considering the speed with which a virus infection becomes established throughout a plant and the high yield of viral-encoded proteins that accumulate in plants, the use of viral vectors provides an attractive and cost effective means for the overproduction of valuable proteins in plants and for rapid evaluation of new traits.
Plant virus-based vectors have been recently developed to express heterologous proteins in plants for the study of gene function, production of pharmaceuticals, analysis of plant-microbe interactions, fungicide and insecticide screening, metabolic engineering and nutrient improvement and represent valuable means to supplement conventional breeding and transgenic technology.
Several different types of positive sense RNA plant viruses have been developed as vectors for production of recombinant proteins and peptides (Pogue et al., Annu. Rev. Phytopathol. 40:45-74 (2002); Scholthof et al., Annu. Rev. Phytopathol. 34:299-323 (1996)). Depending on the structure of the viruses involved and their genome replication and expression strategies, a number of approaches including gene replacement, gene insertion, epitope presentation, and complementation have been utilized. Plant viral vectors are presently available for recombinant protein expression in a wide range of host plants including Nicotiana benthamiana, tobacco, squash, cucumber, wheat, barley, cowpea, Nicotiana clevelandii, Chenopodium quinoa, and Arabidopsis (Allison et al., J. Virol. 62:3581-3588 (1998); Brisson et al., Nature 310:511-514 (1984); Choi et al., Plant J. 23:547-555 (2000); Constantin et al., Plant J. 40:622-631 (2004); Dolja et al., Proc. Natl. Acad. Sci. U.S.A. 89:10208-10212 (1992); Fernandez-Fernandez et al., Virology 280:283-291 (2001); French et al., Science 231:1294-1297 (1986); Gopinath et al., Virology 267:159-173 (2000); Hagiwara et al., J. Virol. 73:7988-7993 (1999); Haupt et al., Plant Physiol. 125:209-218 (2001); Lacomme et al., Plant J. 34:543-553 (2003); Turnage et al., Plant J. 30:107-117 (2002)). Even with these advances, there are only a limited number of plant viral vectors that are suitable for systemic expression of foreign proteins in major legume crops like soybean. Soybean is a main source of oil and high-quality protein worldwide, and there is critical need for tools that allow for rapid evaluation of new traits involving expression of valuable proteins that confer disease/pest resistance and/or those that enhance the commercial value of soybean.
Plant viral vectors can be also used as virus-induced gene silencing (VIGS) reverse genetics tools to study gene function (Burch-Smith et al., 2004). VIGS can specifically down regulate a single gene, members of a gene family, or sets of distinct genes (Lu et al., 2003; Peele et al., 2001; Turnage et al., 2002). Due to these advantages, many positive sense RNA plant viruses have been developed as vectors for production of recombinant proteins or as VIGS vectors for many plant species (Burch-Smith et al., 2004; Ding et al., 2006; Groønlund et al., 2008; Igarashi et al., 2009; Meng et al., 2009; Pogue et al., 2002; Zhang et al., 2009). With readily increasing genomic information, VIGS vectors have substantial potential to advance functional genomics for both monocots and dicots. Methods to understand and analyze plant gene function are employed by using loss-of-function or gain-of-function techniques at present. Gene function analyzed by gain-of-function is generally accomplished through gene transformation, while loss-of-function is conducted through mutagen, transposon tagging, T-DNA insertion or homologous recombination. However, the abovementioned approaches are complicated, time-consuming and difficult to scale up the gene analysis.
Alternatively, gene silencing is recently used to analyze gene function. Virus induced gene silencing (VIGS) is an efficient and reliable method though there are many techniques being conducted to induce gene silencing. Gene transformation is not required in the VIGS process, which provides a quick and feasible way for plants with lengthy life cycle and transformation difficulties. VIGS is an RNA silencing process that exploits a host defense mechanisms to defend against foreign viral RNAs or transposon(s). Small interfering RNA (siRNA) has been observed in plants when foreign viruses invade plants. These siRNAs binds foreign viral RNAs and trigger degradation of viral RNA. Virus-induced gene silencing (VIGS) is a type of RNA silencing that is initiated by recombinant virus vectors carrying fragments of host genes being analyzed. The plants are infected with the recombinant viruses to activate the RNA silencing of endogenous target gene of host plants. Virus-induced gene silencing (VIGS) is an RNA silencing process that targets host defense against viruses. Many plant VIGS vectors have been developed as reverse genetics tools for functional genomics studies. VIGS is especially useful for plants, such as soybean, that are recalcitrant to transformation.
Depending on the genome replication and expression strategy of the virus employed, there are two primary approaches for foreign gene sequence expression by plant viruses. The first is through insertion of foreign gene sequences into the viral genome by substitution of a viral gene or fusion with a viral gene(s) to express the foreign gene as a fusion protein precursor. The second approach is to insert the foreign gene after a viral sub-genomic promoter and express the foreign gene sequence through a viral sub-genomic RNA.
VIGS does not require the whole gene. It was reported that 23 nucleotides was enough to induce gene silencing (Plant J. 25, 417-25, 2001). The use of small fragments alleviates problems of acquiring the whole cDNA and can enhance the specificity of virus induced gene silencing (VIGS). Compared with transformation of plants with sense and/or antisense gene approaches, the advantage of VIGS is the relative speed. Moreover it suppresses the target gene RNA level after the seedling is established, which allows the functions of the essential genes to be tested upon silencing.
Previously, two generations of Bean pod mottle virus (BPMV, genus Comovirus) VIGS vectors have been demonstrated to be effective and efficient soybean functional genomics tools. However, there are critical limitations to the design of the previous vectors. For example, the fragment of the host gene to be silenced must be expressed as a fusion protein that is in the same reading frame as the viral polyprotein. The expression of this peptide may be undesirable for VIGS applications as this feature limits design of VIGS inserts to translatable regions rather than to any region of interest. Further, in the previous design, only one gene can be expressed.
It is an object of the present invention to provide a new DNA-based BPMV vector to facilitate applications of the BPMV vector for soybean functional genomics.
It is yet another object of the present invention to provide a novel BPMV-based vector which accommodates simultaneous expression of foreign genes as well as insertion of non-coding sequences for VIGS.
It is yet another object of the present invention to provide a plant VIGS RNA1 helper vector that has ideal symptom phenotype that does not interfere with the VIGS phenotype.
It is yet a further object of the invention to provide a novel vector for over-expression and accumulation of heterologous proteins in legumes such as soybean as well as for VIGS for loss of function analysis.